Transposase compositions for reduction of insertion bias

ABSTRACT

Presented herein are methods and compositions for tagmentation of nucleic acids. The methods are useful for generating tagged DNA fragments that are qualitatively and quantitatively representative of the target nucleic acids in the sample from which they are generated.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/523,765 filed May 2, 2017 which is the U.S. national phase ofPCT/US2015/059194 filed Nov. 5, 2015 and published in English as WO2016/073690 on May 12, 2016 which claims priority to U.S. Prov. App. No62/075,713 filed on Nov. 5, 2014 and to U.S. Prov. 62/242,935 filed onOct. 16, 2015, which are each hereby incorporated by reference in itsentirety.

REFERENCE TO SEQUENCE LISTING

The present application includes a Sequence Listing in electronicformat. The Sequence Listing is provided as a file entitledILLINC385C1SEQLISTING.TXT, created Sep. 1, 2020, which is approximately1.5 Kb in size. The information in the electronic format of the SequenceListing is incorporated herein by reference in its entirety.

B ACKGROUND

Transposase enzymes are useful in in vitro transposition systems. Theyallow for massive-scale fragmentation and tagging of genomic DNA and areuseful for making libraries of tagged DNA fragments from target DNA foruse in nucleic acid analysis methods such as next-generation sequencingand amplification methods. There remains a need for transposasecompositions and tagmentation methods with improved properties and whichgenerate tagged DNA fragments that are qualitatively and quantitativelyrepresentative of the target nucleic acids in the sample from which theyare generated.

BRIEF SUMMARY

Presented herein are methods and compositions for tagmentation ofnucleic acids. The transposase compositions and tagmentation methodsprovided herein have surprisingly improved properties including, forexample, generating tagged DNA fragments that are qualitatively andquantitatively representative of the target nucleic acids in the samplefrom which they are generated.

Accordingly, one embodiment presented herein is a method of sequentialtagmentation comprising: (a) providing a first transposome, the firsttransposome comprising a first transposase enzyme having a firsttagmentation profile; (b) combining a target nucleic acid with the firsttransposase enzyme under conditions suitable for tagmentation, therebygenerating a tagmented nucleic acid; (c) combining the tagmented nucleicacid with a second transposome under conditions suitable fortagmentation, the transposome comprising a second transposase enzymehaving a tagmentation profile. In some embodiments, the firsttagmentation profile and the second tagmentation profile are different.In some embodiments, the method comprises a wash step between steps (b)and (c) to substantially separate the tagmented nucleic from reactionbuffer used in step (b). In some embodiments, the wash step comprisesbinding the tagmented nucleic acid to a solid support. In someembodiments, the solid support comprises beads. In some embodiments, thesolid support comprises a spin column. In some embodiments, the firsttransposome and the second transposome have different insertion bias. Insome embodiments, step (c) comprises adding the second transposome to areaction mixture comprising the first transposome.

Also presented herein is method of tagmentation comprising: (a)providing a first transposome, the first transposome comprising a firsttransposase enzyme having a first tagmentation profile; (b) providing asecond transposome, the second transposome comprising a secondtransposase enzyme having a second tagmentation profile; (c) combining atarget nucleic acid with the first transposome and the secondtransposome under conditions suitable for tagmentation, therebygenerating a tagmented nucleic acid.

In one aspect, disclosed herein are methods of sequential tagmentation.The methods include providing a first transposome. The first transposomecomprises a first transposase enzyme having a first tagmentationprofile. A target nucleic acid is combined with the first transposaseenzyme under conditions suitable for tagmentation, thereby generating atagmented nucleic acid. The tagmented nucleic acid is combined with asecond transposome under conditions suitable for tagmentation. Thesecond transposome comprises a second transposase enzyme having a secondtagmentation profile.

Embodiment 1. A method of sequential tagmentation comprising: (a)providing a first transposome, the first transposome comprising a firsttransposon and a first transposase enzyme having a first tagmentationprofile; (b) combining a target nucleic acid with the first transposaseenzyme under conditions suitable for tagmentation, thereby generating afirst tagmented nucleic acid; (c) combining the first tagmented nucleicacid with a second transposome under conditions suitable fortagmentation, the transposome comprising a second transposon and asecond transposase enzyme having a second tagmentation profile, therebygenerating a second tagmented nucleic acid.

Embodiment 2. The method of embodiment 1, wherein the first tagmentationprofile and the second tagmentation profile are different.

Embodiment 3. The method of embodiment 1, comprising a wash step betweensteps (b) and (c) to substantially separate the tagmented nucleic fromreaction buffer used in step (b).

Embodiment 4. The method of embodiment 3, wherein the wash stepcomprises binding the tagmented nucleic acid to a solid support.

Embodiment 5. The method of embodiment 4, wherein the solid supportcomprises beads.

Embodiment 6. The method of embodiment 4, wherein the solid supportcomprises a spin column.

Embodiment 7. The method of embodiment 1, wherein the first transposaseand the second transposase have different insertion bias.

Embodiment 8. The method of embodiment 1, wherein step (c) comprisesadding the second transposome to a reaction mixture comprising the firsttransposome.

Embodiment 9. The method of embodiment 1, wherein reaction buffer usedin step (b) is diluted to permit tagmentation reaction with the secondtransposome.

Embodiment 10. A method of preparing a sequencing library, comprising:(a) providing a first transposome, the first transposome comprising afirst transposon and a first transposase enzyme having a firsttagmentation profile, wherein the first transposome is immobilized on afirst solid support; (b) combining a target nucleic acid with the firsttransposase enzyme under conditions suitable for tagmentation, therebygenerating a first tagmented nucleic acid; (c) combining the firsttagmented nucleic acid with a second transposome under conditionssuitable for tagmentation, the transposome comprising a secondtransposon and a second transposase enzyme having a second tagmentationprofile, thereby generating a second tagmented nucleic acid and creatinga sequencing library.

Embodiment 11. The method of embodiment 10, wherein the secondtransposome is immobilized on a second solid support.

Embodiment 12. The method of embodiment 11, wherein the first supportand the second support are different.

Embodiment 13. A method of preparing a sequencing library, comprising:(a) providing a first transposome, the first transposome comprising afirst transposon and a first transposase enzyme having a firsttagmentation profile; (b) combining a target nucleic acid with the firsttransposase enzyme under conditions suitable for tagmentation, therebygenerating a first tagmented nucleic acid; (c) combining the firsttagmented nucleic acid with a second transposome under conditionssuitable for tagmentation, the transposome comprising a secondtransposon and a second transposase enzyme having a second tagmentationprofile, wherein the second transposome is immobilized on a second solidsupport, generating a second tagmented nucleic acid and creating asequencing library.

Embodiment 14. The method of any one of embodiments 10-13, wherein thefirst and the second solid supports are beads.

Embodiment 15. The method of any one of embodiments 1-14, wherein thefirst transposon of the first transposome comprises a first adaptor andthe second transposon of the second transposome comprises a secondadaptor.

Embodiment 16. The method of embodiment 15, wherein the first and secondadaptors comprise a sequence selected from the group consisting ofbarcodes, primer binding sequences, restriction endonuclease sites, andunique molecular indices.

Embodiment 17. The method of embodiment 9, wherein the first, second orboth transposomes are immobilized on solid supports.

Embodiment 18. The method of embodiment 17, wherein the solid support isbead.

Embodiment 19. A method of preparing a sequencing library, comprising:(a) providing a first transposome, the first transposome comprising afirst transposon and a first transposase enzyme having a firsttagmentation profile; (b) combining a target nucleic acid with the firsttransposase enzyme under conditions suitable for tagmentation, therebygenerating a first tagmented nucleic acid; (c) combining the firsttagmented nucleic acid with a second transposome under conditionssuitable for tagmentation, the transposome comprising a secondtransposon and a second transposase enzyme having a second tagmentationprofile, thereby generating a second tagmented nucleic acid; (d)amplifying the second tagmented nucleic acid, thereby creating asequencing library.

Embodiment 20. The method of any one of embodiments 1-19, furthercomprising amplifying the first tagmented nucleic acid.

Embodiment 21. The method of any one of embodiments 1-18, furthercomprising amplifying the second tagmented nucleic acid.

Embodiment 22. The method of embodiment 1-18, further comprisingamplifying the first tagmented and second tagmented nucleic acid.

Embodiment 23. The method of embodiment 19, wherein the first, second orboth transposomes are immobilized on solid supports.

Embodiment 24. The method of embodiment 23, wherein the solid support isbead.

Embodiment 25. A method of preparing a sequencing library, comprising:(a) providing a first transposome, the first transposome comprising afirst transposon and a first transposase enzyme having a firsttagmentation profile; (b) combining a target nucleic acid with the firsttransposase enzyme under conditions suitable for tagmentation, therebygenerating a first tagmented nucleic acid; (c) substantially separatingthe first tagmented nucleic from reaction buffer used in step (b); (d)combining the first tagmented nucleic acid with a second transposomeunder conditions suitable for tagmentation, the second transposomecomprising a second transposon and a second transposase enzyme having asecond tagmentation profile, thereby generating a second tagmentednucleic acid; (e) amplifying the second tagmented nucleic acid, therebygenerating a sequencing library.

Embodiment 26. The method of embodiment 25, further comprisingoptionally amplifying the first tagmented nucleic acid.

Embodiment 27. The method of any one of embodiments 25-26, wherein thefirst, second or both transposomes are immobilized on solid supports.

Embodiment 28. The method of embodiment 27, wherein the solid support isbead.

Embodiment 29. The method of any one of embodiments 19-28, wherein thefirst transposon comprises a first adaptor.

Embodiment 30. The method of any one of embodiments 19-28, wherein thesecond transposon comprises a second adaptor.

Embodiment 31. The method of any one of embodiments 19-28, wherein thefirst transposon comprises a first adaptor and the second transposoncomprises a second adaptor, wherein the first and the second adaptorsare different.

Embodiment 32. The method of any one of embodiments 19-31, wherein thefirst and second adaptors comprise a sequence selected from the groupconsisting of barcodes, primer binding sequences, restrictionendonuclease sites, and unique molecular indices.

Embodiment 33. The method of any one of embodiments 1-32, wherein thefirst transposase enzyme is selected from the group consisting of Mos-1,HyperMu™, Tn5, Ts-Tn5, Ts-Tn5059, Hermes, Tn7.

Embodiment 34. The method of any one of embodiments 1-32, wherein thesecond transposase enzyme is selected from the group consisting ofMos-1, HyperMu™, Tn5, Ts-Tn5, Ts-Tn5059, Hermes, Tn7.

Embodiment 35. The method of any of embodiments 1-34, wherein themethods are used for meta-genomics for microbial samples.

Embodiment 36. The method of any one of embodiments 1-35, wherein thefirst tagmentation profile and the second tagmentation profile aredifferent, and wherein the two profiles have different percent of GCdropout.

Embodiment 37. The method of any one of embodiments 1-35, wherein thefirst tagmentation profile and the second tagmentation profile aredifferent, and wherein the two profiles have different percent of ATdropout.

Embodiment 38. The method of anyone of embodiments 10-37, whereinreaction buffer used in step (b) is diluted to permit tagmentationreaction with the second transposome.

Embodiment 39. The method of anyone of embodiments 10-37, wherein thesecond transposome is combined to a reaction mixture comprising thefirst transposome and the first tagmented nucleic acid.

Embodiment 40. The method of embodiment 39, wherein one or more firsttransposase remains bound to the first tagmented nucleic acid duringcombining the first tagmented nucleic acid with a second transposome.

Embodiment 41. The method of embodiment 8, wherein one or more firsttransposase remains bound to the first tagmented nucleic acid duringcombining the first tagmented nucleic acid with a second transposome.

Embodiment 42. A method of tagmentation comprising: (a) providing afirst transposome, the first transposome comprising a first transposonand a first transposase enzyme having a first tagmentation profile; (b)providing a second transposome, the second transposome comprising asecond transposon and a second transposase enzyme having a secondtagmentation profile; (c) combining a target nucleic acid with the firsttransposome and the second transposome under conditions suitable fortagmentation, thereby generating a tagmented nucleic acid.

Embodiment 43. The method of embodiment 42, wherein the first, second orboth transposomes are immobilized on solid supports.

Embodiment 44. The method of embodiment 43, wherein the solid support isbead.

Embodiment 45. The method of any one of embodiments 42-44, wherein thefirst transposon comprises a first adaptor.

Embodiment 46. The method of any one of embodiments 42-44, wherein thesecond transposon comprises a second adaptor.

Embodiment 47. The method of any one of embodiments 42-44, wherein thefirst transposon comprises a first adaptor and the second transposoncomprises a second adaptor, wherein the first and the second adaptorsare different.

Embodiment 48. The method of any one of embodiments 42-47, wherein thefirst and second adaptors comprise a sequence selected from the groupconsisting of barcodes, primer binding sequences, restrictionendonuclease sites, and unique molecular indices.

Embodiment 49. The method of any one of embodiments 42-48, wherein thefirst transposase enzyme is selected from the group consisting of Mos-1,HyperMu™, Tn5, Ts-Tn5, Ts-Tn5059, Hermes, Tn7.

Embodiment 50. The method of any one of embodiments 42-48, wherein thesecond transposase enzyme is selected from the group consisting ofMos-1, HyperMu™, Tn5, Ts-Tn5, Ts-Tn5059, Hermes, Tn7.

Embodiment 51. The method of any of embodiments 42-50, wherein themethods are used for meta-genomics for microbial samples.

Embodiment 52. The method of any one of embodiments 42-51, wherein thefirst tagmentation profile and the second tagmentation profile aredifferent, and wherein the two profiles have different percent of GCdropout.

Embodiment 53. The method of any one of embodiments 42-51, wherein thefirst tagmentation profile and the second tagmentation profile aredifferent, and wherein the two profiles have different percent of ATdropout.

The details of one or more embodiments are set forth in the accompanyingdrawings and the description below. Other features, objects, andadvantages will be apparent from the description and drawings, and fromthe claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a bar graph of the number of unique molecules in EZ-Tn5™and EZ-Tn5™+Mos1 tagmented DNA libraries prepared using differentconcentrations of transposomes.

FIG. 2 shows a bar graph of the insert size in the tagmented librariesof FIG. 1.

FIG. 3 shows a bar graph of the percent AT dropout and the percent GCdropout in the tagmented libraries of FIG. 1.

FIG. 4 shows a plot of the number of unique molecules in tagmented DNAlibraries prepared using different tagmentation buffer formulations.

FIG. 5 shows a bar graph of the insert size in the tagmented librariesof FIG. 4;

FIG. 6 shows a bar graph of the percent AT dropout and the percent GCdropout in the tagmented libraries of FIG. 4.

FIG. 7 shows a bar graph of the total number of reads and diversity inTS-Tn5059+Mos1 tagmented libraries prepared using differentconcentrations of transposomes.

FIG. 8 shows a bar graph of insert size in the tagmented libraries ofFIG. 7.

FIG. 9 shows a bar graph of the percent AT dropout and the percent GCdropout in the tagmented libraries of FIG. 7.

FIG. 10 shows a plot of the number of unique molecules in Mos 1+Tn5sequentially tagmented DNA libraries.

FIG. 11 shows a plot of the insert size in the tagmented libraries ofFIG. 10;

FIG. 12 shows a plot of the percent AT dropout and the percent GCdropout in the tagmented libraries of FIG. 10.

FIG. 13 shows a bar graph of the number of unique molecules in Mos1+Tn5tagmented DNA libraries prepared using different concentrations oftransposomes; and

FIG. 14 shows a bar graph of the percent AT dropout and the percent GCdropout in the tagmented libraries of FIG. 13.

FIG. 15 shows HyperMu™ transposon sequence (SEQ ID NO:01, and SEQ IDNO:02).

FIG. 16 shows the sequences of HyperMu™ transposon and various primersused including MM1141 (SEQ ID NO:01), MM1138 (SEQ ID NO:02), MuPCRts(SEQ ID NO:03), P7MUTS (SEQ ID NO:04), and P5MUTS (SEQ ID NO:05).

FIG. 17 shows the agarose gel electrophoresis analysis of thefragmentation products of bacteriophage genome at varying concentrationsof HyperMu™ transposome.

FIG. 18 shows the agarose gel electrophoresis analysis of the PCRamplified products of tagmented E. Coli chromosome after tagmentationwith HyperMu™ transposome.

FIG. 19 shows the statistics of a sequencing run of E. Coli chromosomeusing HyperMu™ tagmentation.

FIG. 20 shows the fragment length distribution after tagmentation of E.Coli chromosome using HyperMu™.

FIG. 21 shows the uniformity of coverage of Tn5 alone, HyperMu™ alone,TrueSeq alone, or a combination of HyperMu™ and Tn5.

FIG. 22 A-D show the sequence bias of TruSeq, HyperMu™ and Tn5 (Nextera)and compared to a reference tagmentation. The DNA used in referencestudies is E. coli DNA. % of GC is shown in x-axis and the frequency isshown in y-axis. FIG. 22A is a comparison of the reference withtagmentation results using 3.2 ng of HyperMu™. FIG. 22B is a comparisonof the reference with tagmentation results using 1 ng of Nextera (Tn5)and 3.2 ng of HyperMu™. FIG. 22C is a comparison of the reference withTruSeq method. FIG. 22D is a comparison of the reference with Truseqmethod, tagmentation with Nextera and HyperMu™.

FIG. 23 shows an exemplary scheme of preparing sequence library bysequential tagmentation using HyperMu™ and Nextera transposomes.

FIG. 24 shows a plot of fraction of normalized coverage as a function of% GC using Mos-1 tagmentation followed by TsTn5059/Nextera tagmentation.The experiments include two different workflows, one with a clean-upstep between two tagmentation steps and the other where the buffer isadjusted by dilution between two tagmentation steps.

FIG. 25 shows a plot of fraction of normalized coverage as a function of% GC using TsTn5059/Nextera tagmentation followed by Mos-1 tagmentation.

FIG. 26 shows a plot of coverage uniformity using various combination ofMos-1 and TsTn5059/Nextera enzymes at various concentrations.

FIG. 27 shows a bar graph of the percent AT dropout and the percent GCdropout in the tagmented libraries using various combinations ofTsTn5059/Nextera and Mos-1.

FIG. 28 shows a bar graph showing the diversity inTS-Tn5059/Nextera+Mos1 tagmented libraries prepared using differentconcentrations of transposomes.

DETAILED DESCRIPTION

The transposase compositions and tagmentation methods provided hereinhave surprisingly improved properties including, for example, generatingtagged DNA fragments that are qualitatively and quantitativelyrepresentative of the target nucleic acids in the sample from which theyare generated.

Inventors of the present application have surprisingly and unexpectedlyfound that using two or more transposomes having two or more differenttagmentation profiles provides a more uniform tagmentation of a targetDNA. The inventors have also found that the use of two or moretransposomes is specially advantageous when the tagmentation profiles ofthe transposases have different insertion biases such as different ATand GC dropout rates.

The methods and compositions provided herein are useful with transposaseenzymes and methods as described in greater detail in the disclosure ofU.S. 62/062,006, filed on Oct. 9, 2014 and entitled “MODIFIEDTRANSPOSASES FOR REDUCTION OF INSERTION BIAS,” the content of which isincorporated by reference herein in its entirety.

The methods and compositions provided herein are also useful withtransposase enzymes and methods as described in greater detail in thedisclosures of U.S. 2010/0120098 and 2014/0194324, the content of whichis incorporated by reference herein in its entirety.

In one aspect, the methods disclosed herein include sequentialtagmentation comprising: (a) providing a first transposome, the firsttransposome comprising a first transposon and a first transposase enzymehaving a first tagmentation profile; (b) combining a target nucleic acidwith the first transposase enzyme under conditions suitable fortagmentation, thereby generating a first tagmented nucleic acid; (c)combining the first tagmented nucleic acid with a second transposomeunder conditions suitable for tagmentation, the transposome comprising asecond transposon and a second transposase enzyme having a secondtagmentation profile, thereby generating a second tagmented nucleicacid.

In some embodiments, the method comprises a wash step between steps (b)and (c) to substantially separate the tagmented nucleic from reactionbuffer used in step (b). In some embodiments, the wash step comprisesbinding the tagmented nucleic acid to a solid support. In someembodiments, the solid support comprises beads. In some embodiments, thesolid support comprises a spin column. In some embodiments, step (c)comprises adding the second transposome to a reaction mixture comprisingthe first transposome. In some embodiments, one or more firsttransposases remained bound to the first tagmented nucleic acid duringthe combination of second transposome to the first tagmented nucleicacid.

In one aspect, disclosed herein are methods of preparing a sequencinglibrary. The methods include providing a first transposome, the firsttransposome comprising a first transposon and a first transposase enzymehaving a first tagmentation profile, in which the first transposome isimmobilized on a first solid support. A target nucleic acid is combinedwith the first transposase enzyme under conditions suitable fortagmentation, thereby generating a first tagmented nucleic acid. Thetagmented nucleic acid is combined with a second transposome underconditions suitable for tagmentation, the second transposome comprisinga second transposon and a second transposase enzyme having a secondtagmentation profile, thereby generating a second tagmented nucleic acidand creating a sequencing library.

In one aspect, disclosed herein are methods of preparing a sequencinglibrary. The methods include providing a first transposome, the firsttransposome comprising a first transposon and a first transposase enzymehaving a first tagmentation profile. A target nucleic acid is combinedwith the first transposase enzyme under conditions suitable fortagmentation, thereby generating a first tagmented nucleic acid. Thefirst tagmented nucleic acid is combined with a second transposome underconditions suitable for tagmentation, the second transposome comprisinga second transposon and a second transposase enzyme having a secondtagmentation profile, in which the second transposome is immobilized ona second solid support, thereby generating a second tagmented nucleicacid and creating a sequencing library.

In one aspect, disclosed herein are methods of preparing a sequencinglibrary. The methods include providing a first transposome, the firsttransposome comprising a first transposon and a first transposase enzymehaving a first tagmentation profile. A target nucleic acid is combinedwith the first transposase enzyme under conditions suitable fortagmentation, thereby generating a first tagmented nucleic acid. Thefirst tagmented nucleic acid is combined with a second transposome underconditions suitable for tagmentation, the transposome comprising asecond transposon and a second transposase enzyme having a secondtagmentation profile, thereby creating a sequencing library.

In one aspect, disclosed herein are methods of preparing a sequencinglibrary. The methods include providing a first transposome, the firsttransposome comprising a first transposon and a first transposase enzymehaving a first tagmentation profile. A target nucleic acid is combinedwith the first transposase enzyme under conditions suitable fortagmentation, thereby generating a first tagmented nucleic acid. Thefirst tagmented nucleic acid is substantially separated from thereaction buffer used for the first tagmentation reaction. The firsttagmented nucleic acid is then combined with a second transposome underconditions suitable for tagmentation, the second transposome comprisinga second transposons and a second transposase enzyme having a secondtagmentation profile, thereby generating a second tagmented nucleicacid. The second tagmented nucleic acid is amplified, thereby generatinga sequencing library. In some embodiments, the first set of tagmentednucleic acid is optionally amplified.

In another aspect, the methods disclosed herein include tagmentationcomprising: (a) providing a first transposome, the first transposomecomprising a first transposon and a first transposase enzyme having afirst tagmentation profile; (b) providing a second transposome, thesecond transposome comprising a second transposon and a secondtransposase enzyme having a second tagmentation profile; (c) combining atarget nucleic acid with the first transposome and the secondtransposome under conditions suitable for tagmentation, therebygenerating a tagmented nucleic acid. In some embodiments, the first andsecond transposomes are added simultaneously.

In some embodiments, the first transposon of the first transposomecomprises first adaptor and the second transposon of the secondtransposome comprises a second adaptor. In some embodiments, the firstand the second adaptors are different. In some embodiments, the firstand second adaptors comprise a sequence selected from the groupconsisting of barcodes, primer binding sequences, restrictionendonuclease sites, and unique molecular indices.

In some embodiments, the first, second, or both transposomes areimmobilized on a second solid support. Exemplary solid supports include,but are not limited to beads, flow cell surface, spin column, columnmatrix. In some embodiments, the first surface and the second supportsare different. In some embodiments, the first and the second solidsupports are beads. In some embodiments, the first trasposomes areimmobilized on a solid support. In some embodiments, the firsttransposomes immobilized on a solid support remain bound to thetagmented target nucleic acid and the first tagmented nucleic acids areseparated from the solution using the solid support (e.g., streptavidinbeads, magnetic beads etc.).

In some embodiments of the above aspects, the first tagmented nucleicacid is amplified before contacting the second transposome. In someembodiments of the above aspects, the second tagmented nucleic acid isamplified. In some embodiments, two sets of amplification are carriedout. In the first set of amplification, the first tagmented nucleic acidis amplified. In the second set of amplification, the second tagmentednucleic acid is further amplified.

In some embodiments, the first tagmentation profile and the secondtagmentation profile are different. In some embodiments, the firsttransposase and the second transposase have different insertion bias. Insome embodiments, the first tagmentation and second profiles havedifferent AT dropout rates. In some embodiments, the first and secondtagmentation profiles have different GC dropout rates. In someembodiments, the first tagmentation profile has higher GC dropout rateas compared to the second tagmentation profile. In some embodiments, thefirst transposase has a greater insertion bias towards AT rich region ascompared to the second transposase. In some embodiments, the secondtagmentation profile has higher AT dropout rate as compared to the firsttagmentation profile. In some embodiments, the second transposase has agreater insertion bias towards GC rich region as compared to the firsttransposase.

In some embodiments, the first tagmentation profile has higher ATdropout rate as compared to the second tagmentation profile. In someembodiments, the first transposase has a greater insertion bias towardsGC rich region as compared to the second transposase. In someembodiments, the second tagmentation profile has higher GC dropout rateas compared to the first tagmentation profile. In some embodiments, thesecond transposase has a greater insertion bias towards AT rich regionas compared to the second transposase.

In some embodiments, the reaction buffer used for the first tagmentationreaction is diluted to permit tagmentation reaction with the secondtransposome. In some embodiments, the reaction buffer used for the firsttagmentation reaction is diluted to at least 1.5-fold, 2-fold, 2.5-fold,3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold,12.5-fold, 15-fold, 20-fold, 25-fold, 30-fold, 40-fold, 50-fold,60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 125-fold, 200-fold,250-fold, 300-fold or more. In some embodiments, the first tagmentednucleic acid remains immobilized on a solid support during the dilution.In some embodiments, the first tagmented nucleic acid remains bound toone or more first transposases during the dilution. In some embodiments,the first tagmented nucleic acid remains bound to one or more firsttransposomes and the first transposomes are immobilized on a solidsupport, thereby immobilizing the first tagmented nucleic acid.

In some embodiments, the first and second transposomes have differenttagmentation profile for a particular genome.

In some embodiments, the first transposase for the first tagmentationreaction is Mos-1 and the second transposase for the second tagmentationreaction is Tn5 transposase (e.g., EZTn5™, NexteraV2, or TS-Tn5059).

In one embodiment, the first transposase for the first tagmentationreaction is Tn5 transposase (e.g., EZTn5™, NexteraV2, or TS-Tn5059) anda second transposase for the second tagmentation reaction is Mos1transposases to generate a tagmented DNA library. In one example, thesecond tagmentation reaction using Mos1 is performed immediately afterthe first tagmentation reaction using Tn5 (i.e., a clean-up step is notused to remove Tn5 from the DNA before the second tagmentationreaction).

In another embodiment, the methods of the invention use a firsttagmentation reaction using Mos1 transposases followed by a sampleclean-up step and a second tagmentation reaction using Tn5 transposases(e.g., EZTn5™, NexteraV2, or TS-Tn5059) to generate a tagmented DNAlibrary. In one example, the sample clean-up step is a DNA clean-up stepperformed using the DNA Clean & Concentrator™ kit (Zymo Research). Inthis clean-up step, Mos1 is denatured and removed from the DNA. Inanother example, the sample clean-up step is performed using AgencourtAMPure beads (Beckman Coulter, Inc.). In this example, the clean-up stepis a buffer exchange step wherein the Mos1 transposomes remain bound tothe DNA.

In another embodiment, the methods of the invention use a firsttagmentation reaction using Mu or HyperMu™ transposases followed by asample clean-up step and a second tagmentation reaction using Tn5transposases (e.g., EZTn5™, NexteraV2, or TS-Tn5059) to generate atagmented DNA library. In one example, the sample clean-up step is a DNAclean-up step performed using the DNA Clean & Concentrator™ kit (ZymoResearch). In this clean-up step, Mu or HyperMu™ is denatured andremoved from the DNA. In another example, the sample clean-up step isperformed using Agencourt AMPure beads (Beckman Coulter, Inc.). In thisexample, the clean-up step is a buffer exchange step wherein the Mu orHyperMu™ transposases remain bound to the DNA. FIG. 23 shows anexemplary scheme of preparing sequence library by sequentialtagmentation using HyperMu™ and Nextera transposomes. The input DNA isfirst subjected to tagmentation using the individually tagged“Mu-Nextera” complex. This library is then amplified via PCR, so thatevery fragment is represented multiple times in the amplified reaction.The amplified library is taken through a 2nd round of tagmentation withthe basic Nextera kit in a concentration regime in which the enzyme isthe limiting factor. This guarantees that every single molecule isminimally tagmented with the Nextera complex to preserve maximumcontinuity information. The generated library is then sequenced. All theNextera- fragments that have been generated from the same amplicon doshare the same barcode (or end-tag), which can be used in the finalassembly.

In some embodiments, the amount of first and the second transposases canbe same. In some embodiments, the amount of first and the secondtransposases can be different.

In one embodiment, the methods are used for meta-genomics usingmicrobial samples.

Sequential Tagmentation to Meta-Genomics and Microbiome

Sequential tagmentation in all its forms can be applied to microbialsamples for meta-genomics applications. In some examples, theperformance of a single transposase is highly affected by its bias andthe sequence context of the target DNA. If the sample contains multiplespecies with different DNA sequence compositions, the tagmentation mayperform better for some than the other. Genomic DNA for some species mayend up with relatively larger or much smaller fragments, which skewstheir representation on the flowcell. This will cause missing ofmeta-genomics information. In extreme cases, some species may not haveany representation on the flowcell.

Using sequential tagmentation can help lowering the overall tagmentationbias. In particular, different ratios of the two enzymes can be appliedto tweak the library preparation for various genomic compositions.Multiple sequential tagmentations with different enzyme ratios can beapplied to the same sample. This helps capturing wider range of speciesin a microbiome sample. For example, the sample can be split intomultiple smaller samples and apply sequential tagmentations withdifferent enzyme ratios on each. Different ratios of enzymes can helpbetter capturing sequencing data of different species in the microbiomesample.

Furthermore, a quick screen can be established based on fragment sizedistribution. A microbiome sample (from a certain source, such as gut)can be split into smaller size samples, multiple sequentialtagmentations with different enzyme ratios can be applied to each andfragment size distribution for each one can be stored as the baseline.Sample from the same source can be put through the same process andmajor changes in the fragment size distributions can point towards amajor change in the microbiome. This can be used for a quick test to seewhether a microbiome flora from a source is changing.

As used herein, the term “tagmentation” refers to the modification ofDNA by a transposome complex comprising transposase enzyme andtransposon end sequence in which the transposon end sequence furthercomprises adaptor sequence. Tagmentation results in the simultaneousfragmentation of the DNA and ligation of the adaptors to the 5′ ends ofboth strands of duplex fragments.

Following a purification step to remove the transposase enzyme,additional sequences can be added to the ends of the adapted fragments,for example by PCR, ligation, or any other suitable methodology known tothose of skill in the art.

The method of the invention can use any transposase that can accept atransposase end sequence and fragment a target nucleic acid, attaching atransferred end, but not a non-transferred end. A “transposome” iscomprised of at least a transposase enzyme and a transposase recognitionsite. In some such systems, termed “transposomes”, the transposase canform a functional complex with a transposon recognition site that iscapable of catalyzing a transposition reaction. The transposase orintegrase may bind to the transposase recognition site and insert thetransposase recognition site into a target nucleic acid in a processsometimes termed “tagmentation”. In some such insertion events, onestrand of the transposase recognition site may be transferred into thetarget nucleic acid.

In standard sample preparation methods, each template contains anadaptor at either end of the insert and often a number of steps arerequired to both modify the DNA or RNA and to purify the desiredproducts of the modification reactions. These steps are performed insolution prior to the addition of the adapted fragments to a flowcellwhere they are coupled to the surface by a primer extension reactionthat copies the hybridized fragment onto the end of a primer covalentlyattached to the surface. These ‘seeding’ templates then give rise tomonoclonal clusters of copied templates through several cycles ofamplification.

The number of steps required to transform DNA into adaptor-modifiedtemplates in solution ready for cluster formation and sequencing can beminimized by the use of transposase mediated fragmentation and tagging.

In some embodiments, transposon based technology can be utilized forfragmenting DNA, for example as exemplified in the workflow for Nextera™DNA sample preparation kits (Illumina, Inc.) wherein genomic DNA can befragmented by an engineered transposome that simultaneously fragmentsand tags input DNA (“tagmentation”) thereby creating a population offragmented nucleic acid molecules which comprise unique adaptersequences at the ends of the fragments.

Some embodiments can include the use of a hyperactive Tn5 transposaseand a Tn5-type transposase recognition site (Goryshin and Reznikoff, J.Biol. Chem., 273:7367 (1998)), or MuA transposase and a Mu transposaserecognition site comprising R1 and R2 end sequences (Mizuuchi, K., Cell,35: 785, 1983; Savilahti, H, et al., EMBO J., 14: 4893, 1995). Anexemplary transposase recognition site that forms a complex with ahyperactive Tn5 transposase (e.g., EZ-Tn5™ Transposase, EpicentreBiotechnologies, Madison, Wis.).

More examples of transposition systems that can be used with certainembodiments provided herein include Staphylococcus aureus Tn552 (Colegioet al., J. Bacteriol., 183: 2384-8, 2001; Kirby C et al., Mol.Microbiol., 43: 173-86, 2002), Ty1 (Devine & Boeke, Nucleic Acids Res.,22: 3765-72, 1994 and International Publication WO 95/23875), TransposonTn7 (Craig, N L, Science. 271: 1512, 1996; Craig, N L, Review in: CurrTop Microbiol Immunol., 204:27-48, 1996), Tn/O and IS10 (Kleckner N, etal., Curr Top Microbiol Immunol., 204:49-82, 1996), Mariner transposase(Lampe D J, et al., EMBO J., 15: 5470-9, 1996), Tc1 (Plasterk R H, Curr.Topics Microbiol. Immunol., 204: 125-43, 1996), P Element (Gloor, G B,Methods Mol. Biol., 260: 97-114, 2004), Tn3 (Ichikawa & Ohtsubo, J Biol.Chem. 265:18829-32, 1990), bacterial insertion sequences (Ohtsubo &Sekine, Curr. Top. Microbiol. Immunol. 204: 1-26, 1996), retroviruses(Brown, et al., Proc Natl Acad Sci USA, 86:2525-9, 1989), andretrotransposon of yeast (Boeke & Corces, Annu Rev Microbiol. 43:403-34,1989). More examples include ISS, Tn10, Tn903, IS911, and engineeredversions of transposase family enzymes (Zhang et al., (2009) PLoS Genet.5:e1000689. Epub 2009 Oct. 16; Wilson C. et al (2007) J. Microbiol.Methods 71:332-5). Additionally, the methods and compositions providedherein are useful with transposase of Vibrio species, including Vibrioharveyi, as set forth in greater detail in the disclosures of US2014/0093916 and 2012/0301925, each of which is incorporated byreference in its entirety.

Briefly, a “transposition reaction” is a reaction wherein one or moretransposons are inserted into target nucleic acids at random sites oralmost random sites. Essential components in a transposition reactionare a transposase and DNA oligonucleotides that exhibit the nucleotidesequences of a transposon, including the transferred transposon sequenceand its complement (i.e., the non- transferred transposon end sequence)as well as other components needed to form a functional transposition ortransposome complex. The DNA oligonucleotides can further compriseadditional sequences (e.g., adaptor or primer sequences) as needed ordesired.

The adapters that are added to the 5′ and/or 3′ end of a nucleic acidcan comprise a universal sequence. A universal sequence is a region ofnucleotide sequence that is common to, i.e., shared by, two or morenucleic acid molecules. Optionally, the two or more nucleic acidmolecules also have regions of sequence differences. Thus, for example,the 5′ adapters can comprise identical or universal nucleic acidsequences and the 3′ adapters can comprise identical or universalsequences. A universal sequence that may be present in different membersof a plurality of nucleic acid molecules can allow the replication oramplification of multiple different sequences using a single universalprimer that is complementary to the universal sequence.

As used herein the term “at least a portion” and/or grammaticalequivalents thereof can refer to any fraction of a whole amount. Forexample, “at least a portion” can refer to at least about 1%, 2%, 3%,4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 99.9% or 100% of awhole amount.

As used herein the term “about” means +/−10%.

Solid Support

In some embodiments, the solid support or its surface is non-planar,such as the inner or outer surface of a tube or vessel. In someembodiments, the solid support is a surface of a flow cell. In someembodiments, the solid support comprises microspheres or beads. By“microspheres” or “beads” or “particles” or grammatical equivalentsherein is meant small discrete particles. Suitable bead compositionsinclude, but are not limited to, plastics, ceramics, glass, polystyrene,methylstyrene, acrylic polymers, paramagnetic materials, thoria sol,carbon graphite, titanium dioxide, latex or cross-linked dextrans suchas Sepharose, cellulose, nylon, cross-linked micelles and Teflon, aswell as any other materials outlined herein for solid supports may allbe used. “Microsphere Detection Guide” from Bangs Laboratories, FishersInd. is a helpful guide. In certain embodiments, the microspheres aremagnetic microspheres or beads. In some embodiments, the beads can becolor coded. For example, MicroPlex® Microspheres from Luminex, Austin,Tex. may be used.

The beads need not be spherical; irregular particles may be used.Alternatively or additionally, the beads may be porous. The bead sizesrange from nanometers, i.e. 100 nm, to millimeters, i.e. 1 mm, withbeads from about 0.2 micron to about 200 microns being preferred, andfrom about 0.5 to about 5 micron being particularly preferred, althoughin some embodiments smaller or larger beads may be used. In someembodiments, beads can be about 1, 1.5, 2, 2.5, 2.8, 3, 3.5, 4, 4.5, 5,5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 15, or 20 μm in diameter.

Barcodes

Generally, a barcode can include one or more nucleotide sequences thatcan be used to identify one or more particular nucleic acids. Thebarcode can be an artificial sequence, or can be a naturally occurringsequence generated during transposition, such as identical flankinggenomic DNA sequences (g-codes) at the end of formerly juxtaposed DNAfragments. In some embodiments, a barcode is an artificial sequence thatis non-natural to the target nucleic acid and is used to identify thetarget nucleic acid or determine the contiguity information of thetarget nucleic acid.

A barcode can comprise at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20 or more consecutive nucleotides. Insome embodiments, a barcode comprises at least about 10, 20, 30, 40, 50,60, 70 80, 90, 100 or more consecutive nucleotides. In some embodiments,at least a portion of the barcodes in a population of nucleic acidscomprising barcodes is different. In some embodiments, at least about10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% of the barcodesare different. In more such embodiments, all of the barcodes aredifferent. The diversity of different barcodes in a population ofnucleic acids comprising barcodes can be randomly generated ornon-randomly generated.

In some embodiments, a transposon sequence comprises at least onebarcode. In some embodiments, such as transposomes comprising twonon-contiguous transposon sequences, the first transposon sequencecomprises a first barcode, and the second transposon sequence comprisesa second barcode. In some embodiments, a transposon sequence comprises abarcode comprising a first barcode sequence and a second barcodesequence. In some of the foregoing embodiments, the first barcodesequence can be identified or designated to be paired with the secondbarcode sequence. For example, a known first barcode sequence can beknown to be paired with a known second barcode sequence using areference table comprising a plurality of first and second bar codesequences known to be paired to one another.

In another example, the first barcode sequence can comprise the samesequence as the second barcode sequence. In another example, the firstbarcode sequence can comprise the reverse complement of the secondbarcode sequence. In some embodiments, the first barcode sequence andthe second barcode sequence are different. The first and second barcodesequences may comprise a bi-code.

In some embodiments of compositions and methods described herein,barcodes are used in the preparation of template nucleic acids. As willbe understood, the vast number of available barcodes permits eachtemplate nucleic acid molecule to comprise a unique identification.Unique identification of each molecule in a mixture of template nucleicacids can be used in several applications. For example, uniquelyidentified molecules can be applied to identify individual nucleic acidmolecules, in samples having multiple chromosomes, in genomes, in cells,in cell types, in cell disease states, and in species, for example, inhaplotype sequencing, in parental allele discrimination, in metagenomicssequencing, and in sample sequencing of a genome.

Exemplary barcode sequences include, but are not limited to TATAGCCT,ATAGAGGC, CCTATCCT, GGCTCTGA, AGGCGAAG, TAATCTTA, CAGGACGT, andGTACTGAC.

Target Nucleic Acids

A target nucleic acid can include any nucleic acid of interest. Targetnucleic acids can include DNA, RNA, peptide nucleic acid, morpholinonucleic acid, locked nucleic acid, glycol nucleic acid, threose nucleicacid, mixed samples of nucleic acids, polyploidy DNA (i.e., plant DNA),mixtures thereof, and hybrids thereof. In a preferred embodiment,genomic DNA or amplified copies thereof are used as the target nucleicacid. In another preferred embodiment, cDNA, mitochondrial DNA orchloroplast DNA is used.

A target nucleic acid can comprise any nucleotide sequence. In someembodiments, the target nucleic acid comprises homopolymer sequences. Atarget nucleic acid can also include repeat sequences. Repeat sequencescan be any of a variety of lengths including, for example, 2, 5, 10, 20,30, 40, 50, 100, 250, 500 or 1000 nucleotides or more. Repeat sequencescan be repeated, either contiguously or non-contiguously, any of avariety of times including, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15or 20 times or more.

Some embodiments described herein can utilize a single target nucleicacid. Other embodiments can utilize a plurality of target nucleic acids.In such embodiments, a plurality of target nucleic acids can include aplurality of the same target nucleic acids, a plurality of differenttarget nucleic acids where some target nucleic acids are the same, or aplurality of target nucleic acids where all target nucleic acids aredifferent. Embodiments that utilize a plurality of target nucleic acidscan be carried out in multiplex formats so that reagents are deliveredsimultaneously to the target nucleic acids, for example, in one or morechambers or on an array surface. In some embodiments, the plurality oftarget nucleic acids can include substantially all of a particularorganism's genome. The plurality of target nucleic acids can include atleast a portion of a particular organism's genome including, forexample, at least about 1%, 5%, 10%, 25%, 50%, 75%, 80%, 85%, 90%, 95%,or 99% of the genome. In particular embodiments the portion can have anupper limit that is at most about 1%, 5%, 10%, 25%, 50%, 75%, 80%, 85%,90%, 95%, or 99% of the genome

Target nucleic acids can be obtained from any source. For example,target nucleic acids may be prepared from nucleic acid moleculesobtained from a single organism or from populations of nucleic acidmolecules obtained from natural sources that include one or moreorganisms. Sources of nucleic acid molecules include, but are notlimited to, organelles, cells, tissues, organs, organisms, single cell,or a single organelle. Cells that may be used as sources of targetnucleic acid molecules may be prokaryotic (bacterial cells, for example,Escherichia, Bacillus, Serratia, Salmonella, Staphylococcus,Streptococcus, Clostridium, Chlamydia, Neisseria, Treponema, Mycoplasma,Borrelia, Legionella, Pseudomonas, Mycobacterium, Helicobacter, Erwinia,Agrobacterium, Rhizobium, and Streptomyces genera); archeaon, such ascrenarchaeota, nanoarchaeota or euryarchaeotia; or eukaryotic such asfungi, (for example, yeasts), plants, protozoans and other parasites,and animals (including insects (for example, Drosophila spp.), nematodes(e.g., Caenorhabditis elegans), and mammals (for example, rat, mouse,monkey, non-human primate and human). Target nucleic acids and templatenucleic acids can be enriched for certain sequences of interest usingvarious methods well known in the art. Examples of such methods areprovided in Int. Pub. No. WO/2012/108864, which is incorporated hereinby reference in its entirety. In some embodiments, nucleic acids may befurther enriched during methods of preparing template libraries. Forexample, nucleic acids may be enriched for certain sequences, beforeinsertion of transposomes after insertion of transposomes and/or afteramplification of nucleic acids.

In addition, in some embodiments, target nucleic acids and/or templatenucleic acids can be highly purified, for example, nucleic acids can beat least about 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% free fromcontaminants before use with the methods provided herein. In someembodiments, it is beneficial to use methods known in the art thatmaintain the quality and size of the target nucleic acid, for exampleisolation and/or direct transposition of target DNA may be performedusing agarose plugs. Transposition can also be performed directly incells, with population of cells, lysates, and non-purified DNA.

In some embodiments, target nucleic acid can be from a single cell. Insome embodiments, target nucleic acid can be from formalin fixedparaffin embedded (FFPE) tissue sample. In some embodiments, targetnucleic acid can be cross-linked nucleic acid. In some embodiments, thetarget nucleic acid can be cross-linked to nucleic acid. In someembodiments, the target nucleic acid can be cross-linked to proteins. Insome embodiments, the target nucleic acid can be cell-free nucleic acid.Exemplary cell-free nucleic acid include but are not limited tocell-free DNA, cell-free tumor DNA, cell-free RNA, and cell-free tumorRNA.

In some embodiments, target nucleic acid may be obtained from abiological sample or a patient sample. The term “biological sample” or“patient sample” as used herein includes samples such as tissues andbodily fluids. “Bodily fluids” may include, but are not limited to,blood, serum, plasma, saliva, cerebral spinal fluid, pleural fluid,tears, lactal duct fluid, lymph, sputum, urine, amniotic fluid, andsemen. A sample may include a bodily fluid that is “acellular.” An“acellular bodily fluid” includes less than about 1% (w/w) wholecellular material. Plasma and serum are examples of acellular bodilyfluids. A sample may include a specimen of natural or synthetic origin(i.e., a cellular sample made to be acellular).

The term “Plasma” as used herein refers to acellular fluid found inblood. “Plasma” may be obtained from blood by removing whole cellularmaterial from blood by methods known in the art (e.g., centrifugation,filtration, and the like).

Methods of Use

The transposases presented herein can be used in a sequencing procedure,such as an in vitro transposition technique. Briefly, in vitrotransposition can be initiated by contacting a transposome complex and atarget DNA. Exemplary transposition procedures and systems that can bereadily adapted for use with the transposases of the present disclosureare described, for example, in WO 10/048605; US 2012/0301925; US2013/0143774, each of which is incorporated herein by reference in itsentirety.

For example, in some embodiments, the transposase enzymes presentedherein can be used in a method for generating a library of tagged DNAfragments from target DNA comprising any dsDNA of interest (e.g., foruse as next-generation sequencing or amplification templates), themethod comprising: incubating the target DNA in an in vitrotransposition reaction with at least one transposase and a transposonend composition with which the transposase forms a transpositioncomplex, the transposon end composition comprising (i) a transferredstrand that exhibits a transferred transposon end sequence and,optionally, an additional sequence 5′-of the transferred transposon endsequence, and (ii) a non-transferred strand that exhibits a sequencethat is complementary to the transferred transposon end sequence, underconditions and for sufficient time wherein multiple insertions into thetarget DNA occur, each of which results in joining of a first tagcomprising or consisting of the transferred strand to the 5′ end of anucleotide in the target DNA, thereby fragmenting the target DNA andgenerating a population of annealed 5′-tagged DNA fragments, each ofwhich has the first tag on the 5′-end; and then joining the 3′-ends ofthe 5′-tagged DNA fragments to the first tag or to a second tag, therebygenerating a library of tagged DNA fragments (e.g., comprising eithertagged circular ssDNA fragments or 5′- and 3′-tagged DNA fragments (or“di-tagged DNA fragments”)).

As used herein the term “diversity” refers to the number of uniquemolecules in a library. In some embodiments, diversity is an indicationof the diversity (complexity) of the library.

As used herein “insert size” means average fragment size for thelibrary. The mode and mean insert sizes were determined based onsequencing data, after determining the length of the sequenced insert.In some embodiments, fragment size is determined using a BioAnalyzer.

As used herein “GC dropout” means the percentage of GC rich regions inthe genome that are dropped (absent) from the tagmented library.

As used herein “AT dropout” means the percentage of AT rich regions inthe genome that are dropped (absent) from the tagmented library.

As used herein, the term “insertion bias” refers to the sequencepreference of a transposase for insertion sites. For example, if thebackground frequency of A/T/C/G in a polynucleotide sample is equallydistributed (25% A, 25% T, 25% C, 25% G), then any over-representationof one nucleotide over the other three at a transposase binding site orcleavage site reflects an insertion bias at that site. Insertion biascan be measured using any one of a number of methods known in the art.For example, the insertion sites can be sequenced and the relativeabundance of any particular nucleotide at each position in an insertionsite can be compared.

Unless otherwise specified, the terms “a” or “an” mean “one or more”throughout this application.

EXAMPLES Example 1 Tn5 and Mos1 Sequential Tagmentation Tn5 TagmentationFollowed by Mos1 Tagmentation

In some experiments, first tagmentation reaction using Tn5 transposomes(e.g., EZTn5™, NexteraV2, or TS-Tn5059) and a second tagmentationreaction using Mos1 transposomes to generate a tagmented DNA library. Inone example, the second tagmentation reaction using Mos1 is performedimmediately after the first tagmentation reaction using Tn5 (i.e., aclean-up step is not used to remove Tn5 from the DNA before the secondtagmentation reaction). The Tn5 enzyme used was either EZTn5, Tn5 fromNextera V2 kit, or mutant TS-Tn5059.

To evaluate the effect of sequential tagmentation using Tn5 and Mos1transposomes on library output and sequencing metrics, tagmented DNAlibraries were constructed using Bacillus cereus genomic DNA. For eachsequentially tagmented library, a first tagmentation reaction wasperformed by mixing 20 μL B. cereus genomic DNA (50 ng), 25 μL 2×standard tagmentation buffer (2× TD; 20 mM Tris Acetate, pH 7.6, 10 mMMgCl2, and 20% dimethylformamide (DMF)), and various concentrations ofEZ-Tn5™ transposomes (Epicentre) in a total reaction volume of 50 μL.EZ-Tn5™ transposome was used at final concentrations of 3, 6, 12, 25,50, and 100 nM. Reactions were incubated at 55° C. for 5 minutes. Aftercompletion of the first tagmentation reaction, a second tagmentationreaction using Mos1 transposomes was performed. Mos1 transposome wasused at final concentrations of 20 and 100 nM. Reactions using Mos1 alsoincluded the addition of NaCl at a final concentration of 200 mM.Reactions were incubated at 30° C. for 60 minutes.

For each Tn5 control library, a tagmentation reaction was performed bymixing 20 μL B. cereus genomic DNA, 25 μL 2× standard tagmentationbuffer, and 5 μL of EZ-Tn5™ (25 nM) or NexteraV2 (25 nM) transposomes ina total reaction volume of 50 μL. Reactions were incubated at 55° C. for5 minutes.

Following the tagmentation reaction, the samples were processedaccording to the rest of the standard Nextera™ sample preparationprotocol (after tagmentation reaction) Libraries were sequenced bysequencing-by-synthesis (SBS) and evaluated by standard next generationsequencing analysis tools. Fragment size distribution in each librarywas also evaluated on a Bioanalyzer.

FIG. 1 shows a bar graph 100 of the number of unique molecules inEZ-Tn5™ and EZ-Tn5™+Mos1 tagmented DNA libraries prepared usingdifferent concentrations of transposomes. The number of unique moleculesin a library is an indication of the diversity (complexity) of thelibrary. Each bar on the graph represents a tagmented library. Controllibraries (i.e., libraries that were prepared using standard reactionconditions of 25 nM EZ-Tn5™ or NexteraV2 transposomes) are designated by“std”. Libraries that were prepared using different concentrations ofEZ-Tn5™ are designated by “EZTn5—enzyme concentration”. For example, thethird bar in bar graph 100 is labeled “EZTn5-100 nM” and designates alibrary that was prepared using EZ-Tn5™ at a final concentration of 100nM. Libraries that were prepared using sequential tagmentation withEZ-Tn5™ followed by Mos1 are designated by “EZTn513 enzymeconcentration—Mos1—enzyme concentration”. For example, the ninth bar inbar graph 100 is labeled “EZTNS-100 nM-Mos1-20 nM” and designates alibrary that was prepared using EZ-Tn5™ at a final concentration of 100nM followed by tagmentation using Most at a final concentration of 20nM. All libraries were prepared using the standard buffer formulation. Aline 110 indicates a standard level of diversity obtained in a Tn5tagmented DNA library.

The data show that tagmented libraries prepared using EZ-Tn5™ at 12nM-50 nM and Mos1 sequential tagmentation at 20 nM have a higher averagediversity compared to control libraries and libraries prepared usingEZ-Tn5™ alone. For example, the “EZTn5-25 nM-Mos1-20 nM” library hasabout a two-fold increase in diversity compared to control libraries orlibraries prepared using EZ-Tn5™ alone.

FIG. 2 shows a bar graph 200 of the insert size in the tagmentedlibraries of FIG. 1. The median value and mode value for each tagmentedlibrary were generated from the SBS data. Because the median and modevalues were generated from the SBS data, only those inserts that wereamplified in the cluster amplification process and sequenced arerepresented. The “BA” value was generated from a Bioanalyzer trace ofthe fragment size distribution in each tagmented sample. Because the“BA” value was generated from the Bioanalyzer trace, all fragmentsgenerated in the tagmentation reaction are represented (i.e., largerfragments that may not be represented in the SBS data). The data showthat there is variability in the insert size in libraries prepared usingdifferent concentrations of transposomes. The data also shows that inthe “EZTn5-25 nM-Mos1-20 nM”, which is the library with the highestlevel of diversity (FIG. 1), the insert size is about the same as theinsert size in the “EZTn5-25 nM” library.

FIG. 3 shows a bar graph 300 of the percent AT dropout and the percentGC dropout in the tagmented libraries of FIG. 1. AT dropout may bedefined as the percentage of AT rich regions in the genome that are notpresent in the tagmented library. GC dropout may be defined as thepercentage of GC rich regions in the genome that are not present in thetagmented library. A line 310 indicates a standard threshold of ATdropout obtained in a standard Tn5 tagmentation reaction. In a standardTn5 tagmentation reaction, GC dropout is not typically observed. Thedata show that there is variability in the percent AT and GC dropoutprepared using different transposome concentrations. The data also showsthat in the “EZTn5-25 nM-Mos1-20 nM”, which is the library with thehighest level of diversity (FIG. 1), the percent AT and GC dropout issubstantially lower than the percent dropout in the control libraries,e.g., “EZTn5-std-bcereus”, “NexteraV2-std-bcereus”, and “EZTn5-25 nM”.

To evaluate the effect of tagmentation buffer composition on libraryoutput and sequencing metrics, B. cereus tagmented libraries wereprepared using modified formulations of the standard tagmentationbuffer. The buffer formulations were as follows standard buffer (TD) (10mM Tris Acetate, pH 7.6, 5 mM MgCl2, and 10% DMF); manganese buffer (Mn;10 mM Tris Acetate, pH 7.6, 5 mM MnCl2, and 10% DMF); cobalt buffer (Co;10 mM Tris Acetate, pH 7.6, 5 mM CoCl2, and 10% DMF); and nickel buffer(Ni; 10 mM Tris Acetate, pH 7.6, 5 mM NiCl2, and 10% DMF).

FIG. 4 shows a plot 400 of the number of unique molecules in tagmentedDNA libraries prepared using different tagmentation buffer formulations.Control libraries that were prepared using the standard tagmentationbuffer and volume of transposomes (5 μL=25 nM transposome) aredesignated “EZTn5-std-bcereus” and “NexteraV2-std- bcereus”. Librariesthat were prepared using different volumes (i.e., 10 μL or 15 μL; or 50nM and 75 nM, respectively) of NexteraV2 transposome and the standardtagmentation buffer formulation are designated by “NexteraV2-10 μL” and“NexteraV2-15 μL”. NexterV2 libraries that were prepared using amodified (e.g., Mn, Co, or Ni) tagmentation buffer formulation aredesignated by “NexteraV2-modification” or a “NexteraV2-modification-μL”,where “μL” designates the volume of transposome used in the reaction.Libraries that were prepared using sequential tagmentation withNexteraV2 (25 nM) and Mos1 are designated by “NexteraV2-MBPMos1-20nM-Mn”, where 20 nM is the concentration of Mos1 transposome and “Mn” isthe manganese buffer formulation. The sequential tagmentation libraryreaction was repeated three times (n=3) and the individual libraries aredesignated as a, b or c.

The data show that NexteraV2-tagmented libraries prepared using atagmentation buffer that includes CoCl2 have a higher diversity comparedto libraries prepared in buffers without the addition of CoCl2 (i.e.,the standard tagmentation buffer and buffers that include MnCl2 orNiCl2). The data also shows that libraries prepared using sequentialtagmentation with NexteraV2 and Mos1 in Mn buffer have a higherdiversity compared to tagmented libraries prepared with NexteraV2 alonein Mn buffer.

FIG. 5 shows a bar graph 500 of the insert size in the tagmentedlibraries of FIG. 4. The data show that insert size inNexteraV2-tagmented libraries prepared using tagmentation buffers thatinclude either CoCl2 or NiCl2 are relatively larger compared totagmented libraries prepared using the standard or Mn bufferformulations. The data also show that in the “NexteraV2-MBPMos1-20nM-Mn” sequentially tagmented libraries the insert size is about thesame as the NexteraV2 control library (“NexteraV2-std-bcereus”) andNexteraV2 library prepared in Mn buffer (“NexteraV2-Mn”).

Referring now to FIG. 4 and FIG. 5, the data also shows that insequentially-tagmented libraries prepared using the Mn bufferformulation (NexteraV2-MBPMos1-20 nM-Mn), library diversity is increased(relative to control levels) while the insert size in the libraryremains about the same (relative to control levels).

FIG. 6 shows a bar graph 600 of the percent AT dropout and the percentGC dropout in the tagmented libraries of FIG. 4. The data show that inthe libraries prepared using Mn buffer and sequential tagmentation withNexterV2 and Mos1 (i.e., “EZTn5-25 nM-Mos1-20 nM”) there is asubstantial decrease in the percent AT dropout and a slight increase inthe percent GC dropout compared to libraries prepared using NexteraV2alone and either the standard tagmentation buffer (i.e.,“NexteraV2-std-bcereus”) or Mn buffer (“NexteraV2-Mn).

Referring now to FIGS. 4 through 6, the data shows that in thesequentially-tagmented libraries prepared using the Mn bufferformulation (NexteraV2-MBPMos1-20 nM-Mn), library diversity (FIG. 4) isincreased while the insert size (FIG. 5) in the library remains aboutthe same, and the percent AT dropout (FIG. 6) is substantially reduced.

In another example, the Tn5 transposome TS-Tn5059 and Mos1 were used togenerate sequentially-tagmented B. cereus libraries. In this example, afirst tagmentation reaction was performed using TS-TN5059 at finalconcentrations of 40 nM, 80 nM, and 240 nM. A second tagmentationreaction was performed using Mos1 at final concentrations of 10 and 20nM. All libraries were prepared using the standard buffer formulation.Reactions using Mos1 also included the addition of NaCl at a finalconcentration of 200 mM. Following the tagmentation reaction, thesamples were processed according to the standard Nextera™ samplepreparation protocol. Libraries were evaluated by SBS on a MiSeqinstrument (Illumina, Inc.). Fragment size distribution in each librarywas also evaluated on a Bioanalyzer.

FIG. 7 shows a bar graph 700 of the total number of reads and diversityin TS-Tn5059+Mos1 tagmented libraries prepared using differentconcentrations of transposomes. The total number of reads is the totalnumber of reads from the flow cell. The diversity is the number ofunique molecules in the library and is used as an indication of librarycomplexity. Each pair of bars on the graph represents a tagmentedlibrary. Libraries that were prepared using sequential tagmentation withTS-Tn5059 and Mos1 are designated by “TS-Tn5059—enzymeconcentration—Mos1—enzyme concentration”. EZTn5™ and NexteraV2 were usedto prepare comparative control libraries (e.g., “EZTn5-std-bcereus” and“NexteraV2-std-bcereus”). “NexteraV2-SSB-bcereus” designates a librarythat was prepared using the standard volume (5 μL) of transposome, butincluded an additional 10 μl of SSB diluent. “NexteraV2-15 μL-bcereus”designates a library that was prepared using 15 μL (75 nM) of NexterV2transposome. All libraries were prepared using the standard bufferformulation.

The data shows that the diversity in the TS-Tn5059-240 nM-Mos1-20 nMlibrary is higher compared to the diversity in the EZTn5™ and NexteraV2control libraries.

FIG. 8 shows a bar graph 800 of insert size in the tagmented librariesof FIG. 7. This graph shows the effect of Mos1 concentration to thefinal insert sizes. A Mos1 concentration of the range 10-20 nM does nothave a major impact on the insert sizes. FIG. 9 shows a bar graph 900 ofthe percent AT dropout and the percent GC dropout in the tagmentedlibraries of FIG. 7. The data show that in general the librariesprepared using sequential tagmentation there is a substantial decreasein the percent AT dropout and a slight increase in the percent GCdropout compared to the NexteraV2 control libraries. For example, thepercent AT dropout in the TS-Tn5059-40 nM-Mos1-20 nM is substantiallydecreased and the percent GC dropout slightly increased compared to theNexteraV2-std-bcereus library.

Example 2

Generating Sequencing Library with Mu Transposome Complex

HyperMu™ (Epicentre, Madison, Wis.), a mutant Mu transposase is used togenerate sequencing library. HyperMu™ Transposase, a hyperactive enzymethat retains the highly random insertion characteristics of MuAtransposase but is at least 50-times more active in vitro than theenzyme available from other suppliers. Upon Mu transposition , thetransposon arms (i.e. RI and R11) together with any attached sequencewould be transferred to the template DNA, at the same time fragmentingthe template. HyperMu™ transposon sequence and the primer sequences areshown in FIG. 15-16. The Tn5 enzyme used was EZTn5, Tn5 from Nextera V2kit.

Assessing Activity of Mu Transposome

The tagmentation capacity of HyperMu™ transposome is assessed on a ˜50kb bacteriophage genome. The tagmentation reaction was carried out in TAbuffer for one hour at 37° C. at increasing concentration of HyperMu™transposome. The fragmentation products were analyzed by agarose gelelectrophoresis and the results are shown in FIG. 17.

The HyperMu™ complexes were used to tagment E. coli chromosome and thetagmented fragments were amplified to introduce sequencing adapters. 25cycles of PCR were carried out using P5-MUTS and P7-MUTS primers. ThePCR products are analyzed by agarose gel electrophoresis and shown inFIG. 18. PCR products were observed with different amounts of input DNA.

Analysis of PCR Amplified Tagmented DNA

A 3.2 ng DNA was used for paired end sequencing on GAIIX (2X35 bp). Thesample was sequenced on a single lane on GA. The total number of readswas 30,071,951 and number of unique reads <2000 bp was 4,599,874. Thestatistics of the sequencing run is shown in FIG. 19. The average sizeof the fragments sequenced was much longer than Nextera, and was closeto 800bp as shown in FIG. 20.

The uniformity of the sequencing run was better for HyperMu™ compared toTn5. The uniformity of the sequencing run was better for HyperMu™ withTn5 as compared to HyperMu™ or Tn5 alone. The uniformity of thesequencing run was comparable to TruSeq. The results are shown in FIG.21. The observed GC bias for HyperMu™ is similar to Tn5 and shown inFIG. 22.

The experiments set forth in FIGS. 1-5 were performed to characterizethe effect of Tn5 tagmentation buffer composition and reactionconditions on library output and sequencing metrics. In particular, inexperiments set forth in FIGS. 1-3, Mos-1 was added to Tn5 tagmentationreactions. The Tn5 enzyme used was EZTn5, Tn5 from Nextera V2 kit, ormutant TS-Tn5059.

In experiments set forth in FIGS. 4 and 5, Mos-1 tagmentation reactionwas performed first, and the tagmented DNA was washed to removetagmentation buffer from the first reaction. Subsequently, the tagmentedDNA was further tagmented using a Tn5 enzyme (EZTn5, Tn5 from Nextera V2kit, or mutant TS-Tn5059). Various wash methods tested included Ampurebeads and Zymo Clean and Concentrator kit (Zymo).

To evaluate the effect of tagmentation buffer composition and reactionconditions on library output and sequencing metrics, Tn5 tagmented DNAlibraries were constructed using Bacillus cereus genomic DNA. Eachtagmented library was prepared using 25 ng input of B. cereus genomicDNA. The genomic content of B. cereus is about 40% GC and about 60% AT.

Tagmentation buffers were prepared as 2× formulations. For each library,a tagmentation reaction was performed by mixing 20 μL B. cereus genomicDNA (25 ng), 25 μL 2× tagmentation buffer, and 5 μL enzyme (10×Ts-Tn5059 or 10× Ts-Tn5) in a total reaction volume of 50 μL. Reactionswere incubated at 55° C. for 5 minutes. Following the tagmentationreaction, the samples were processed according to the standard Nextera™sample preparation protocol. Libraries were sequenced using Illumina'sSBS (sequencing-by-synthesis) chemistry on a MiSeq device. Sequencingruns were 2×71 cycles using a V2 MiSeq kit. Fragment size distributionin each library was evaluated on a Bioanalyzer.

Example 3 Tn5 and Mos1 Sequential Tagmentation in Solution

Tn5 transposases have decreased activity in buffers that contain >50 mMNaCl and Mos1 transposases have decreased activity in buffers thatcontain <150 mM NaCl. A sequential tagmentation to optimize the activityof both enzyme can be carried out.

Tn5 Tagmentation Followed by Mos1 Tagmentation

Standard Nextera™ (Epicentre, Madison, Wis.) tagmentation usingconditions and buffers specified in the Nextera library prep protocolsare used. After the initial Tn5 tagmentation, the reaction temperatureis reduced to <30° C. Concentrated NaCl is then added to the reaction toa final concentration of 150-300mM. Mos1 transposome is then added tothe tagmentation reaction and the reaction is incubated at 30° C. Oncetagmentation is complete, the reaction is cleaned-up either usingAmpure™ beads (Beckman Coulter, Calif., USA) or Zymo Clean &Concentrator™ cartridges (Zymo Research, Irvine, Calif.). The remainingprotocol for adaptor addition through PCR and final clean-up and sizeselection is specified in the Nextera™ library prep protocol.

Mos1 Tagmentation Followed by Clean-Up to Remove the High Salt Buffer,Followed by Tn5 Tagmentation

Standard Nextera™ (Epicentre, Madison, Wis.) buffers are used with theinclusion of 150-300 mM NaCl for Mos1 optimal activity. Mos1 is thenadded to the reaction and incubated at 30° C. Post tagmentation, thereaction is cleaned-up using Ampure™ beads (Beckman Coulter, Calif.,USA) or Zymo Clean & Concentrator™ cartridges (Zymo Research, Irvine,Calif.), or similar methods for DNA purification. The cleaned-upfragmented DNA material is then used as the DNA input for the secondarytagmentation using Tn5 or Tn5 mutant transposomes. After the secondtagmentation, the reaction is cleaned-up either using Ampure™ beads(Beckman Coulter, Calif., USA) or Zymo Clean & Concentrator™ cartridges(Zymo Research, Irvine, Calif.), or similar methods. The remainingprotocol for adaptor addition through PCR and final clean-up and sizeselection is specified in the Nextera™ library prep protocol (Epicentre,Madison, Wis).

Mos1 Tagmentation Followed by a Dilution to Reduce the High Salt Buffer,Followed by Tn5 Tagmentation

Standard Nextera™ buffers (Epicentre, Madison, Wis.) are used with theinclusion of 150-300 mM NaCl for Mos1 optimal activity. Mos1 is thenadded to the reaction and incubated at 30° C. Post tagmentation, thereaction is diluted with reaction buffer to reduce the NaClconcentration to ≤50 mM. Tn5 is then added to the tagmentation reactionand incubated at ≤55° C. After the second tagmentation, the reaction isclean-up either using Ampure™ beads (Beckman Coulter, Calif., USA) orZymo Clean & Concentrator™ cartridges (Zymo Research, Irvine, Calif.),or similar DNA purification methods. The remaining protocol for adaptoraddition through PCR and final clean-up and size selection is specifiedin the Nextera library prep protocol™ (Epicentre, Madison, Wis.).

Throughout this application various publications, patents and/or patentapplications have been referenced. The disclosure of these publicationsin their entireties is hereby incorporated by reference in thisapplication.

The term comprising is intended herein to be open-ended, including notonly the recited elements, but further encompassing any additionalelements.

A number of embodiments have been described. Nevertheless, it will beunderstood that various modifications may be made. Accordingly, otherembodiments are within the scope of the following claims.

1.-53. (canceled)
 54. A method for preparing a nucleic acid library,comprising: (a) contacting a target nucleic acid with a firsttransposome in a reaction buffer, thereby generating a first pluralityof tagmented nucleic acids; (b) contacting the first plurality oftagmented nucleic acids with a second transposome to generate a secondplurality of tagmented nucleic acids; and (c) amplifying the secondplurality of tagmented nucleic acids to generate a nucleic acid libraryhaving an increased representation of different sequences of the targetnucleic acid compared to a nucleic acid library prepared by a singletagmentation of the target nucleic acid.
 55. The method of claim 54,wherein the nucleic acid library comprises an increased representationof A/T-rich genomic sequences or G/C-rich genomic sequences compared toa nucleic acid library prepared by a single tagmentation of the targetnucleic acid.
 56. The method of claim 54, wherein the first transposomeand the second transposome each comprise a different insertion bias intoa nucleic acid.
 57. The method of claim 54, wherein the firsttransposome comprises a transposase selected from a Tn5 transposase, aMu transposase, and a Mos-1 transposase; and the second transposomecomprises a transposase selected from a Tn5 transposase, a Mutransposase, and a Mos-1 transposase.
 58. The method of claim 54,wherein the reaction buffer comprises a Mn cation.
 59. The method ofclaim 54, wherein the first transposome comprises a transposoncomprising a first barcode and a second barcode indicative of acontinuity of tagmented nucleic acids in the first plurality oftagmented nucleic acids.
 60. The method of claim 54, further comprisingamplifying the first plurality of tagmented nucleic acids prior to step(b).
 61. The method of claim 54, wherein the first transposome isimmobilized on a first solid support.
 62. The method of claim 61,wherein the first transposome is removed from the reaction buffer priorto step (b).
 63. The method of claim 62, wherein the first plurality oftagmented nucleic acids are attached to the first transposome.
 64. Themethod of claim 54, wherein the second transposome is immobilized on asecond solid support.
 65. The method of claim 54, wherein the firstplurality of tagmented nucleic acids is removed from the reaction bufferprior to step (b).
 66. The method of claim 54, wherein a saltconcentration of the reaction buffer is adjusted prior to step (b). 67.The method of claim 66, wherein the salt concentration of the reactionbuffer is adjusted by dilution of the reaction buffer.
 68. The method ofclaim 66, wherein the salt concentration of the reaction buffer isadjusted by addition of salt to the reaction buffer.
 69. The method ofclaim 54, wherein the target nucleic acid comprises genomic DNA.
 70. Themethod of claim 54, wherein an amount of first transposome and an amountof the second transposome have a ratio in a range from 1:0.4 to 1:1.7.71. A method for preparing a nucleic acid library having an increasedrepresentation of A/T-rich or G/C-rich sequences, comprising: (a)generating a first plurality of tagmented nucleic acids by contacting atarget nucleic acid with a first transposome; (b) generating a secondplurality of tagmented nucleic acids by contacting the first pluralityof tagmented nucleic acids with a second transposome; and (c) amplifyingthe second plurality of tagmented nucleic acids to generate a nucleicacid library.
 72. The method of claim 71, wherein the first transposomecomprises a transposase selected from a Tn5 transposase, a Mutransposase, and a Mos-1 transposase; and the second transposomecomprises a transposase selected from a Tn5 transposase, a Mutransposase, and a Mos-1 transposase.
 73. The method of claim 71,wherein the reaction buffer comprises a Mn cation.